Objective: Sampledrawing is an important procedure in the study of allergic airway inflammation.The authors investigate the methods of drawing samples from the animals with allergic airway inflammation.Methods: We included in this study 20 Guinea pigs,10 rats and 20 mice,which underwent trachea incision,followed by bronchoalveolar and rhinal lavage and collection of the lavage fluids.Then we collected blood samples via the heart from the guinea pigs and rats and via both the heart and the eyes from the mice,and obtained the tissues of the nasal cavities and lungs by different methods.Results: All the samples were satisfactorily obtained from the animals,and 80% of the bronchoalveolar and rhinal lavage fluids were collected.Conclusion: Different methods should be adopted to suit different sampledrawing from the animal models of allergic airway inflammation.

Objective To develop and validate a method for detecting factor 8 gene (F8) mutations in hemophilia A patients by Ion Torrent semiconductor sequencing .Methods Intron 22 and intron 1 inversions of F8 gene were identified by long distance PCR (LD-PCR), other mutations in the F8 gene were identified by Ion Torrent sequencing.Candidate variants were validated by Sanger sequencing .Sanger sequencing was applied to screen HA carriers from 11 female family members in the 8 pedigrees.One pregnant woman was offered prenatal diagnosis via analyzing the fetal DNA obtained through amniocentesis . Results Four missense mutations ( c.1331A >C, 1648C >T, c.6506G >A, c.6544C >T), two frameshift mutations ( c.2393 _2394insT, c.6320delG), one splicing mutation ( IVS5 +5G >A), one nonsense mutation (c.43C >T) and one Inv22 mutation were identified in all nine probands respectively . Among 11 female family members, 10 females were identified to be HA carriers, and one didn′t carry the maternal pathogenic mutation.Prenatal diagnosis result showed that the fetus inherited the wild -type maternal allele and was predicted to be unaffected by HA .Conclusion The targeted Ion Torrent sequencing is a reliable and efficient method to detect F8 mutations in patients with Hemophilia A disease .

Objectives To study the mutation spectrum in CYP21A2 gene in patients with 21-hydroxylase deficiency (21-OHD), and to analyze the relationship between genotype and phenotype. Methods Eighteen patients with 21-OHD were identified by neonatal screening of 17α-OH progesterone (17α-OHP). The allele specific PCR-DNA sequencing com-bining with multiplex ligation-dependent probe amplification was applied to determine the genotype in the patients and their parents. Results Six mutations of CYP21A2 gene were identified. I2G (44.4%) and del (33.3%) were the most frequent mutations and also were the most common mutations in salt-wasting form. The detection rate of I172N mutation in simple virilizing form was 75%. Patients were classified into three groups according to the degree of 21-hydroxylase enzymatic compromise caused by the mutation. The serum 17α-OHP, ACTH and T levels which reflected the severity of disease were significantly different among three groups (P<0.05). Conclusions The genetic diagnosis of 21-OHD reveals the consistency between genotype and phenotype.

Child ; Chromosome Duplication ; genetics ; Craniofacial Abnormalities ; diagnosis ; genetics ; Facies ; Genetic Diseases, X-Linked ; diagnosis ; genetics ; Humans ; Intellectual Disability ; diagnosis ; genetics ; Male ; Methyl-CpG-Binding Protein 2 ; genetics ; Oligonucleotide Array Sequence Analysis ; Polymorphism, Single Nucleotide ; Sex Chromosome Disorders ; diagnosis ; genetics

OBJECTIVETo analyze two fetuses with multiple malformations revealed by ultrasonography using single nucleotide polymorphism array (SNP array), and to explore the strategy for the prenatal diagnosis of 1p36 deletion syndrome.

METHODSAmniocentesis was performed on the two pregnant women. Amnion fluid cells were cultured, and karyotypes of the fetuses were determined through G-banding analysis. Whole genome SNP array was used to detect genomic anomalies of the two fetuses. The karyotypes of their parents were determined through G-banding analysis of peripheral venous blood samples.

RESULTSG-banding analysis showed a 46,XY,add(1p36)? and a 46,XX,add(1p36)? karyotype for fetuses 1 and 2, respectively. SNP array analysis showed that the fetus 1 had arr[19]1p36.33p36.32 (752 566 - 3 393 462)×1 and 7q35q36.3 (144 480 549 - 159 119 486)×3, and fetus 2 had arr[19]1p36.33p36.23 (752 566 - 8 362 754)×1, 6p25.3p22.3 (204 909 - 20 182 185)×3. The mother of fetus 1 had a 46,XX,t(1;7)(p36;q35) karyotype, and the mother of fetus 2 had a 46,XX,t(1;6)(p36;p22) karyotype. The karyotypes of both fathers appeared to be normal.

CONCLUSIONSNP array has the advantages such as high sensitivity and high accuracy for prenatal diagnosis, and can provide more detailed information for genetic counseling of 1p36 deletion syndrome.

Adult ; Amniocentesis ; Chromosome Banding ; Chromosome Deletion ; Chromosome Disorders ; diagnosis ; Chromosomes, Human, Pair 1 ; Female ; Humans ; Karyotyping ; Polymorphism, Single Nucleotide ; Pregnancy ; Prenatal Diagnosis

OBJECTIVETo explore the value of multiplex ligation-dependent probe amplification (MLPA) for rapid detection of aneuploidies and structural chromosomal abnormalities during prenatal diagnosis.

METHODSTwo hundred and eight six amniotic fluid samples were analyzed with both MLPA and conventional karyotyping. Structural abnormalities were verified with array comparative genomic hybridization.

RESULTSTen cases of trisomy 21, 2 cases of trisomy 18, 1 case of trisomy 13, 1 case of mosaic trisomy 21, 1 case of 45,X, 1 case of large deletion of Xp, 1 case of trisomy 18p and 1 case of large deletion of 18p and 18q were identified. The same results were derived by both MLPA and conventional karyotyping. Structural abnormalities were verified by array comparative genomic hybridization (aCGH) with 100% accuracy.

CONCLUSIONIn addition to aneuploidies, MLPA can rapidly identify large deletions and duplications of chromosomes 21, 18, 13, X and Y. MLPA is supplementary to conventional karyotyping for identification of such chromosomal abnormalities prenatal diagnosis.

Adult ; Aneuploidy ; Female ; Humans ; Multiplex Polymerase Chain Reaction ; methods ; Pregnancy ; Prenatal Diagnosis ; methods ; Young Adult

OBJECTIVETo detect the pathogenic mutation in a patient with methylmalonic acidemia using IonTorrent Personal Genome Machine (PGM) and assess the feasibility of such technology for analyzing complex monogenic diseases.

METHODSPeripheral blood sample was collected from the patient. Genomic DNA was isolated using a standard method and subjected to targeted sequencing using an Ion Ampliseq Inherited Disease Panel. DNA fragment was ligated with a barcoded sequencing adaptor. Template preparation, emulsion PCR, and Ion Sphere Particles enrichment were carried out using the Ion One Touch system. Data from the PGM runs were processed using Ion Torrent Suite 3.2 software to generate sequence reads. All variants were filtered against dbSNPl37. DNA sequences were visualized with an Integrated Genomics Viewer.

RESULTSAfter data analysis and database filtering, a previously reported nonsense mutation, c.586C>T (p.R196X), and a novel mutation c.898C>T (p.R300X) were identified in the MMAA gene in this patient. Both mutations were verified by conventional Sanger sequencing.

CONCLUSIONPathogenic MMAA mutations have been identified in a patient with methylmalonic acidemia. This new-generation targeted sequencing on the PGM sequencers can be applied for genetic diagnosis of hereditary diseases.

Amino Acid Metabolism, Inborn Errors ; genetics ; High-Throughput Nucleotide Sequencing ; methods ; Humans ; Mutation

OBJECTIVETo identify pathogenic mutations in a Chinese pedigree affected with methylmalonic academia for genetic counseling and prenatal diagnosis.

METHODSMolecular analysis of the MUT, MMACHC, MMAA and MMAB genes was performed for the proband with methylmalonic academia by Ion Torrent semiconductor sequencing. Candidate mutations were validated by Sanger sequencing. The couple was offered prenatal diagnosis via analyzing of the fetal DNA through amniocentesis.

RESULTSThe proband was found to be compound heterozygous for c.609G>A (p.Trp203X) and c.658-660del AAG (p.Lys220del) mutations, which were inherited respectively from each of his parents. Prenatal diagnosis showed that the fetus has inherited two wild-type parental alleles.

CONCLUSIONThe targeted Ion Torrent PGM sequencing has detected pathogenic mutations in the Chinese pedigree affected with methylmalonic academia, which has provided molecular evidence for clinical diagnosis, genetic counseling and prenatal diagnosis for the family.

Adult ; Alkyl and Aryl Transferases ; genetics ; Amino Acid Metabolism, Inborn Errors ; embryology ; genetics ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; Carrier Proteins ; genetics ; China ; Female ; High-Throughput Nucleotide Sequencing ; instrumentation ; methods ; Humans ; Infant ; Male ; Methylmalonyl-CoA Mutase ; genetics ; Mitochondrial Membrane Transport Proteins ; genetics ; Molecular Sequence Data ; Mutation ; Pedigree ; Pregnancy ; Prenatal Diagnosis ; instrumentation ; methods

OBJECTIVE: To identify pathogenic mutations in 25 Chinese pedigrees affected with congenital adrenal hyperplasia (CAH). METHODS: Mutations of the CYP21A2 gene were detected with locus-specific PCR/restriction endonuclease analysis, multiplex ligation-dependent probe amplification assay, and direct sequencing of the entire CYP21A2 gene. Prenatal diagnosis was offered to fetuses at risk for CAH. RESULTS: All 50 alleles of the CYP21A2 gene carried by the 25 pedigrees were successfully delineated. Large deletions and conversions have accounted for 16 (32%) of the alleles, which included 9 entire CYP21A2 gene deletions, 6 chimeric CYP21A1P/CYP21A2 genes, and 1 partial conversion of the CYP21A2 gene. For the remaining 34 alleles, there were 9 micro-conversions and 4 de novo mutations [including a previously unreported c.62G>A (p.Trp21X) mutation]. Prenatal diagnosis was provided for 28 fetuses with a high risk for CAH, among whom 8 were found to be affected. CONCLUSION The detection of CYP21A2 gene mutations can facilitate appropriate genetic counseling and prenatal diagnosis for the affected pedigrees.
Adrenal Hyperplasia, Congenital ; diagnosis ; genetics ; Asian Continental Ancestry Group ; China ; Female ; Humans ; Mutation ; Pedigree ; Pregnancy ; Prenatal Diagnosis ; Steroid 21-Hydroxylase ; genetics

Objective:To investigate the relationship between polymorphisms and haplotypes of cyclin-dependent kinase inhibitor 2B antisense RNA 1 ( CDKN2 B- AS1) gene and the risk of ulcerative colitis (UC). Methods:From January 2012 to January 2021, a total of 534 UC patients diagnosed at the Department of Gastroenterology, the Second Affiliated Hospital of Wenzhou Medical University (Yuying Children′s Hospital) and during the same period 560 gender- and age-matched healthy controls were selected. Genotypes of CDKN2 B- AS1 (rs1063192, rs10757274, rs10757278, rs1333048, rs2383207) in venous blood were determined by matrix assisted laser desorption ionization time-of-flight mass spectrometry technique. Unconditional logistic regression was used to analyze the difference in the distribution of CDKN2 B- AS1 gene polymorphisms between UC patients and healthy controls, as well as the influence on the clinicopathologic characteristics of UC patients. Software Haploview 4.2 was used to analyze the linkage disequilibrium and haplotype. Chi-square test was used for statistical analysis. Results:The frequencies of variant genotype (AG+ GG) and variant allele (G) of rs1063192 in UC patients were higher than those in healthy controls (32.4%, 173/534 vs. 24.8%, 139/560; 18.1%, 193/1 068 vs. 13.7%, 153/1 120), and the differences were statistically significant ( OR=1.45 and 1.40, 95% confidence interval(95% CI) 1.12 to 1.89 and 1.11 to 1.77, P=0.006 and 0.004, corrected P=0.030 and 0.020). The frequency of variant allele (G) of rs10757274 in UC patients was lower than that in healthy controls (34.7%, 371/1 068 vs. 39.5%, 442/1 120), and the difference was statistically significant ( OR=0.82, 95% CI 0.69 to 0.98, P=0.025). However, the difference was not significant after Bonferroni correction (corrected P>0.05). According to the Montreal classification, the frequency of homozygous variant genotype (GG) of rs1063192 in the patients with extensive colitis was higher than that in patients with proctitis plus left-sided colitis (6.6%, 14/211 vs. 1.9%, 6/323), and the difference was statistically significant ( OR=3.92, 95% CI 1.47 to 10.42, P=0.006, corrected P=0.030). There was linkage disequilibrium among rs10757274, rs2383207, rs10757278 and rs1333048 of CDKN2 B- AS1 gene. The frequency of haplotype GGGC in UC patients was lower than that in healthy controls (33.3%, 355.5/1 068 vs. 37.8%, 423.4/1 120), and the frequency of haplotype AGGC in UC patients was higher than that in healthy controls (6.7%, 71.7/1 068 vs. 3.6%, 40.3/1 120), and the differences were statistically significant ( χ2=4.81 and 11.16, P=0.028 and<0.001). Conclusions:The variation of rs1063192 in CDKN2 B- AS1 gene may increase the risk of UC. The risk of extensive colitis in patients carrying homozygous variant genotype (GG) of rs1063192 may rise. Among the haplotypes composed of rs10757274, rs2383207, rs10757278 and rs1333048, the risk of UC may decrease in the individuals carrying haplotype GGGC. However, the risk of UC may increase in the individuals carrying haplotype AGGC. The correlation between the variation of 10757274 and the risk of UC still needs to be further verified by expanding the sample size.